Method for preventing and treating crop hemiptera injurious insects by using Bt proteins
A technology for crops and hemiptera, applied in the field of using Bt protein to prevent and control crop hemiptera pests, can solve the problem of no anti-hemiptera insect activity, etc., and achieve the effect of reducing agricultural production costs and reducing yield loss
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Embodiment 1
[0017] Embodiment 1, construct a vector that Agrobacterium transforms rice to express insect-resistant gene
[0018] According to rice codon preference, three insect-resistant protein coding genes, cry30Aa1, cry30Db1 and cry30Ea2, were designed (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1-3, and its 5' end was designed with a BamHI site, 3 The SacI site was designed at the 'end, and the gene fragment can be obtained by double digestion with BamHI-SacI), which was artificially synthesized by Shanghai Sangong. For rice, the rice actin 1 promoter (pOsActin1, Genbank: NC_008398) was selected to initiate the expression of cry30Aa1, cry30Db1, cry30Ea2 genes, and its 5' end was designed with a HindIII site, and its 3' end was designed with a BamHI site. HindIII-BamHI fragment; maize phosphoenolpyruvate carboxylase terminator (PEPC-ter, Genebank: X15239) was selected as the terminator of the gene, the 5' end was designed with a SacI site, and the...
Embodiment 2
[0020] Embodiment 2, the acquisition of transgenic rice
[0021] The method of obtaining transgenic rice is to adopt the existing technology (Lu Xiongbin, Gong Zuxun, 1998 Life Science 10: 125-131; Liu Fan et al., 2003 Molecular Plant Breeding 1: 108-115). Ripe and plump rice seeds were selected for hulling, and callus was induced as transformation material. Get the Agrobacterium containing the target gene (pCambia1300-pOsActin1-cry30Aa1, pCambia1300-pOsActin1-cry30Db1, pCambia1300-pOsActin1-cry30Ea2 prepared in Example 1) to draw a plate, and pick a single clone colony to inoculate and prepare to transform Agrobacterium. Put the rice callus to be transformed into OD 600 In the Agrobacterium liquid (containing acetosyringone) between 0.3-0.4, let the Agrobacterium bind to the surface of the callus, then transfer the callus to the co-culture medium, and co-culture for 2-3 days. Wash the transformed callus with sterile water, transfer to the selection medium containing antibio...
Embodiment 3
[0022] Embodiment 3, the mensuration of insect resistance ability of transgenic crops
[0023] Utilize brown planthopper and white-backed planthopper to measure the insect-resistant activity of the 10 transgenic rice lines obtained in Example 2, the assay method is: the newly hatched nymphs of brown planthopper and white-backed planthopper are connected to transgenic rice and non-transgenic rice in the same growth period For rice, each strain was placed in a separate insect cage, cultured in a culture room with a constant temperature of 25°C and a constant humidity of 85% for 5 to 7 days, and recorded data. Eight of the 10 transgenic lines tested were not harmed by insects, and all the insects died; all insects on the non-transgenic control group survived, and the rice was severely damaged. The mortality rate of brown planthoppers is shown in Table 1.
[0024]Table 1 Mortality of BPH bioassay on transgenic rice
[0025] Genetically modified rice containing insect-re...
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